Muscle-specific splicing of a heterologous exon mediated by a single muscle-specific splicing enhancer from the cardiac troponin T gene - PubMed (original) (raw)

Muscle-specific splicing of a heterologous exon mediated by a single muscle-specific splicing enhancer from the cardiac troponin T gene

T A Cooper. Mol Cell Biol. 1998 Aug.

Abstract

The chicken cardiac troponin T (cTNT) gene contains a single 30-nucleotide alternative exon that is included in embryonic striated muscle and skipped in the adult. Transient-transfection analysis of cTNT minigenes in muscle and fibroblast cell cultures previously identified four muscle-specific splicing enhancers (MSEs) that promote exon inclusion specifically in embryonic striated muscle cultures. Three MSEs located in the intron downstream from the alternative exon were sufficient for muscle-specific exon inclusion. In the present study, the boundaries of these MSEs were defined by scanning mutagenesis, allowing analysis of individual elements in gain-of-function experiments. Concatamers of MSE2 were necessary and sufficient to promote muscle-specific inclusion of a heterologous exon, indicating that it is a target for muscle-specific regulation. Sequences present in MSE2 are also found in MSE4, suggesting that these two MSEs act in a similar manner. MSE3 appears to be different from MSE2 and MSE4 yet is able to functionally replace both of these elements, demonstrating functional redundancy of elements that are likely to bind different factors. MSE2 and MSE4 each contain a novel sequence motif that is found adjacent to a number of alternative exons that undergo regulated splicing in striated muscle, suggesting a common role for this element in muscle-specific regulation.

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Figures

FIG. 1

FIG. 1

cTNT MSEs regulate splicing of a heterologous exon flanked by heterologous splice sites. The diagram illustrates that a 46-nucleotide exon (open box) flanked by the last 99 nucleotides of cTNT intron 4 and the first 142 nucleotides of cTNT intron 5 (thin lines) was inserted into a constitutively spliced minigene derived from skeletal troponin T (thick lines and filled boxes). The relative positions of the oligonucleotides used for RT-PCR are indicated by arrows below the diagram. Nucleotide substitutions within the cTNT splice sites (underlined) were used to introduce the 3′ and 5′ splice sites of human β-globin intron 1. RTB33.51 contains the natural cTNT splice sites. The R35 series constructs contain the globin splice sites flanking an exon of the size indicated in parentheses. Minigenes were transiently transfected into QT35 fibroblasts (F) and primary chicken embryo skeletal muscle (M) cultures and assayed and quantitated as described in Materials and Methods. n, number of independent transfections.

FIG. 2

FIG. 2

Scanning mutagenesis of the MSEs in cTNT intron 5. Shown for each clone are the name, the percent of spliced mRNA that includes the exon in fibroblast (F) and muscle (M) cultures (with standard deviation), the number of transfections (n), and whether splicing is regulated in muscle (REG). Constructs marked with a minus sign do not express statistically significant higher levels of exon inclusion in muscle cultures than in nonmuscle cultures. For example, the difference between the average levels of exon inclusion for construct RTB76-81 is not significant due to the high variability of the results indicated by the large standard deviations. Several mutations alter basal splicing efficiency (see text). The focus of this analysis is the difference in level of exon inclusion between fibroblasts and muscle for each construct. The significance of construct-to-construct variation in the basal level of exon inclusion is less clear since different mutations of the same nucleotides have very different effects on basal splicing efficiency (see Discussion). Nucleotide substitutions are indicated in boldface type. All constructs contain two nucleotide substitutions (positions +71 and +74), which created an _Asp_718 cloning site. Previously defined MSEs are also indicated in boldface type in the genomic sequence. MSEs are redefined based on the results of the scanning mutation analysis presented in this figure. The revised boundaries are indicated by overlining above the genomic sequence. A boundary is defined as the nucleotide adjacent to a mutation that did not affect enhanced splicing in muscle. For MSE2, the 5′ splice site is excluded from the 5′ boundary. The primary goal for defining these boundaries was to identify elements for gain-of-function experiments. MSE2 and MSE3 segments used for gain-of-function studies are underlined in the genomic sequence. na, not applicable.

FIG. 3

FIG. 3

Sequence alignment of MSE2 and MSE4. Matching nucleotides are underlined. A motif in positions 25 to 32 is repeated in positions 33 to 40 and 113 to 121. The sequence in boldface type is found within introns that flank alternative exons that are regulated in muscle (see Fig. 6).

FIG. 4

FIG. 4

Gain-of-function assays for MSE2 and MSE3. MSE2(×3) contains three copies of MSE2 (positions +3 to +46 of intron 5) (Fig. 2) placed 16 nucleotides downstream from the 46-nucleotide heterologous exon. Upstream of the exon are the last 99 nucleotides of cTNT intron 4, which contains MSE1. MSE3(×3) and MSE3(×6) contain three or six copies of MSE3 (positions +57 to +90 of intron 5) (Fig. 2) placed 16 nucleotides downstream of the alternative exon. The MSE3 concatamers in MSE3mut(×6) contain the RTB66-81 mutation (Fig. 2). The percent of spliced mRNA that includes the alternative exon in fibroblasts (F) and muscle (M) is indicated with standard deviation and number of transfections (n). Whether splicing is (+) or is not (−) regulated in muscle (REG) is also indicated.

FIG. 5

FIG. 5

MSE2 is sufficient for muscle-specific exon inclusion. Three copies of MSE2 were placed upstream and downstream of a 45- or 57-nucleotide heterologous exon. The percent of spliced mRNA that includes the alternative exon in fibroblasts (F) and muscle (M) is indicated with standard deviation and number of transfections (n).

FIG. 6

FIG. 6

Sequence comparisons of introns flanking alternative exons that undergo regulated splicing in striated or smooth muscle has identified a novel motif. The position of the motif in MSE2 and MSE4 is indicated by shading in the diagram. The number indicates the position of the first nucleotide of the sequence from the regulated alternative exon. Negative numbers are upstream and positive numbers are downstream from the alternative exon. Abbreviations: αTm, α-tropomyosin; βTm, β-tropomyosin; and sTNT, skeletal troponin T.

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